A well known class of hydrolase enzymes are lipases, of which microbial and fungal cutinases [Purdy et al., Biochem. 14:2831-2840 (1975)] are especially useful for laundry applications because they hydrolyze oily stains and are active in the pH 8-11 range, which is typically employed in laundry solutions.
Lipases are hydrolase enzymes naturally produced by a wide variety of living organisms from microbes to higher eukaryotes. Fatty acids undergoing oxidation in tissues of higher animals must be in free form (that is, non-esterified) before they can undergo activation and oxidation. Thus, intracellular lipases function to hydrolyze the triacylglycerols to yield free fatty acids and glycerol.
Bacterial lipases are classically defined as glycerolesterhydrolases (EC 3.1.1.3) since they are polypeptides capable of cleaving glycerol ester bonds. They have a high affinity for interfaces, a characteristic which distinguishes them from other enzymes such as proteases and esterases. An interface onto which lipases readily absorb is that of oil/water.
Cutinases are hydrolase enzymes that catalyst the hydrolysis of cutin. For example, cutinase allows fungi to penetrate through the cutin barrier into the host plant during the initial stages of a fungal infection. The primary structures of several cutinases have been compared and shown to be strongly conserved. Ettinger, Biochemistry, 26, pp. 7883-7892 (1987). Sebastian et al., Arch. Biochem. Biophys., 263 (1), pp. 77-85 (1988) have recently found production of cutinase to be induced by cutin in a fluorescent P. putida strain. This cutinase catalyzed hydrolysis of p-nitrophenyl esters of C.sub.4 -C.sub.16 fatty acids.
Lipases have long been considered as potential components in detergent compositions. An early preparation of lipase in the form of pancreatin was taught for addition to detergent formulations by Rohm, Chem. Abs., Int., P2048 (1916). More recently, lipases obtained from certain Pseudomonas or Chromobacter microorganisms have been disclosed as useful in detergent compositions: Thom et al., U.S. Pat. No. 4,707,291, issued Nov. 17, 1987 and Wiersema et al., European Patent Application 253,487, published Jan. 20, 1988.
Lipases have long been known generally to be inhibited by anionic detergents and by nonionic detergents. Lipase activity has been reported as enhanced by emulsifiers such by Wills, Bioch., 60, pp. 529-534 (1955) and Andree, et al., J. App. Biochem., 2, pp. 218-229 (1980). Not withstanding such teachings, attempts to use lipases in laundry solutions which contain anionic or nonionic surfactants have been largely unsuccessful, and effective use of lipases for cleaning oily stains have been limited to presoak applications.
U.S. Pat. No. 3,950,277, inventors Stewart et al., issued Apr. 13, 1976 describes pre-soak compositions employing a lipase enzyme and a lipase activator selected from the group consisting of naphthalene sulfonates, certain polyoxylalkylene derivatives of ethylene diamine and certain acylamino acid salts.
Lipases are useful in the absence of surfactant for removing oily stains and soil from fabrics by using them in aqueous solutions for prewash or presoak applications over extended periods of time, followed by a conventional washing with a fully formulated detergent. Under these conditions, lipases are effective in removing natural oil (triglyceride-containing) stains. However, despite many attempts to use lipases commercially in detergent-containing laundering solutions, the demonstrated washing benefit has been disappointing because lipases generally do not function in a predictably satisfactory manner in the presence of surfactants.
Attempts have recently been made to find specific lipases that are less adversely affected by the surfactants in wash solutions. Japanese Patent Application 63039579, published Feb. 20, 1988 states that a novel lipase obtained from a Pseudomonas is only slightly inhibited by anionic surfactants and is activated by non-ionic surfactants.
European Patent Application 258,068, published Mar. 2, 1988 reports a lipase from the genus Thermomyces said to be compatible with anionic surfactants and effective as a detergent additive.
European Patent Application 268,456 discloses an enzymatic peracid bleaching system for in situ generation of peracid, which is useful for laundry bleaching and which employs (a) a novel enzyme having hydrolase activity (isolatable from Pseudomonas putida (ATCC 53552); (b) a hydrolyzable substrate capable of peroxidation, such as a triglyceride, e.g., trioctanoin and tridecanoin; and (c) a source of peroxyoxygen which, by reaction with (a) and (b), produces a peracid. Unlike that system, the present invention is not peracid based and does not require the relatively longer period of time required to form peracid for operability although, like that process, the compound which it employs as an activator for the enzyme can be a triglyceride.
U.S. Pat. No. 4,981,611 states that combinations of a cutinase enzyme and a nonionic, anionic, cationic or zwitteronic surfactant are useful as cleaning agents and that two or more surfactants exhibit a synergistic effect with respect to hydrolysis activity. However, only the combination of Triton X-100 (octoxynol) and SDS (sodium dodecyl sulfate) and the bacterial cutinase from ATCC 53552 was disclosed in support of the latter property and the single claims of that patent is limited to that specific combination. In actual fact, only certain types of surfactants are capable of preventing the inactivating effect of most surfactants.
There is no published explanation for the compatibility or incompatibility of specific lipases with various laundry and cleaning formulations, and notwithstanding the disclosure of general compatibility of cutinase enzymes with surfactants in U.S. Pat. No. 4,981,611, it has been generally recognized in the art that many surfactants when present in conventional concentrations in wash water often partially or completely inhibit the hydrolysis activity of most lipases. As a consequence, extended presoaking of oily soiled or stained fabrics in a lipase solution in the absence of surfactant is required in order to ensure that the desired enzymatic hydrolysis of any oily soil or stains will occur.
We have found that the hydrolysis inhibiting activity of surfactants as a class on the oily soil and stain removing enzymatic activity of hydrolase enzymes generally can be suppressed or eliminated by the presence of an effective amount of a non-charged hydrolysis activator, such as a lipophilic compound, e.g., an oily liquid or semi-solid hydrocarbon, of a natural or synthetic fatty ester, a substantially water insoluble hydrocarbon bearing a polar functional group, and certain non-ionic surfactants, which amount varies from lipase to lipase and from type of surfactant to type of surfactant. With this knowledge, hydrolase-based detergent compositions and laundry additive compositions which are effective in surfactant-containing laundry water and which have effective oily stain and soil removing activity can be formulated.